GC-rich Regions Research Articles

24. Identification of recurrent, non-coding mutations across breast cancer molecular subtypes

Many studies have attempted pan-cancer analyses in order to identify recurrent coding mutations, but few have explored non-coding space, and often the results do not extend to breast cancer. While some breast cancer specific studies have identified significantly mutated promoters, they have often failed to incorporate transcriptome data to assess the impact and relevance of mutations on gene expression within tumors (Nik-Zainal et al. 2016, Rheinbay et al. 2017). In order to address this issue, we have compiled a data set consisting of 458 breast cancer tumor/normal pairs. Mutations have been identified with whole genome, exome, or custom capture sequencing and expression with transcriptome sequencing or microarray. The custom (regulome) capture reagent covers regions assembled from regulatory databases, 5’ untranslated regions, 500 bases upstream and downstream of transcription start sites, and 50,000 bases upstream and downstream of 178 genes that have been implicated as being important in breast cancer (Lesurf et al. 2016). While this custom capture region is similar in size to an exome, it has advantages over WGS and exome sequencing. It provides deep sequence coverage of regulatory regions, including GC-rich promoter regions, where WGS often produces insufficient coverage. After extensive filtering, our preliminary analysis revealed significant mutation clustering within the noncoding space of RMRP, WDR74, as noted in previous studies, as well as ∼50 other genes not previously reported. The significance of these mutations, based on recurrence, transcriptome changes, and molecular subtype relationships will be presented.

Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.

The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with Taq DNA polymerase.

Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte.

The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.

Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks.

Aberrant DNA methylation is a distinguishing feature of cancer. Yet, how methylation affects immune surveillance and tumor metastasis remains ambiguous. We introduce a novel method, Guide Positioning Sequencing (GPS), for precisely detecting whole-genome DNA methylation with cytosine coverage as high as 96% and unbiased coverage of GC-rich and repetitive regions. Systematic comparisons of GPS with whole-genome bisulfite sequencing (WGBS) found that methylation difference between gene body and promoter is an effective predictor of gene expression with a correlation coefficient of 0.67 (GPS) versus 0.33 (WGBS). Moreover, Methylation Boundary Shift (MBS) in promoters or enhancers is capable of modulating expression of genes associated with immunity and tumor metabolism. Furthermore, aberrant DNA methylation results in tissue-specific enhancer switching, which is responsible for altering cell identity during liver cancer development. Altogether, we demonstrate that GPS is a powerful tool with improved accuracy and efficiency over WGBS in simultaneously detecting genome-wide DNA methylation and genomic variation. Using GPS, we show that aberrant DNA methylation is associated with altering cell identity and immune surveillance networks, which may contribute to tumorigenesis and metastasis.

Structure, behaviour and repetitive DNA of B-chromosomes in Cestrum nocturnum (Solanaceae)

The Cestrum genus is karyotypically exceptional in Solanaceae. It is characterised by a basic number x = 8, a large chromosomal and genomic size, complex heterochromatin patterns, B-chromosomes (Bs) with particular heterochromatin and distribution of 18–5.8–26S and 5S rDNA. Cestrum nocturnum L. has a diploid number of 2n = 16 plus a variable number of B-chromosomes. The aims of work was to analyse their numerical variation, structure and behaviour of C. nocturnum B-chromosomes by classical and molecular cytogenetics. The individuals analysed had 2n = 16 + 0−13 B-chromosomes. All B-chromosomes were metacentric and smaller than A-chromosomes. The number of B-chromosomes showed a great variability between and within individuals, thereby denoting the occurrence of events that promote mitotic and meiotic instability. Cytogenetic techniques made it possible to observe that B-chromosomes are rich in heterochromatin, probably with AT- and GC-rich regions. In addition, molecular techniques allowed to detect homologous sequences of transposable element conserved domains of Ty1-Copia and Ty3-Gypsy superfamilies. These sequences were located by FISH in all B-chromosomes and some A-chromosomes. Our results showed that repetitive DNA could play an important role in chromosomal evolution as well as in the stability of B-chromosomes in C. nocturnum.

Depicting the DNA binding and photo-nuclease ability of anti-mycobacterial drug rifampicin: A biophysical and molecular docking perspective

Rifampicin, an important member of ansamycin family, exhibits various biological activities. It is frequently used for the treatment of tuberculosis and leprosy. Recently, its interaction with protein is evidenced. But, its interaction with DNA is still unknown. Whether, exhibition of anti-cancer activity of rifampicin is associated with DNA-cleavage activity is also unknown. In this study, an attempt has been taken to understand these two unknown aspects. Spectroscopic studies indicated that rifampicin binds to CT-DNA with a binding constant of ~5.22 × 105 M−1. Several independent experiments like CD analysis, competitive displacement experiments and viscosity measurements revealed that rifampicin intercalates into the CT-DNA. Molecular docking studies corroborate this fact and depicted that this drug binds to the GC-rich region of DNA through multiple hydrogen bonding having the relative binding energy of −9.21 kcal mol−1. Besides, DNA binding ability, rifampicin causes the photo-cleavage of pUC19 DNA via singlet oxygen pathway. To the best of our knowledge, we report for the first time the DNA binding and DNA cleavage ability of rifampicin. This study provides a clue behind the execution of the anti-cancer activity of rifampicin. Overall, all these information can be used for further understanding the pharmacological effects of rifampicin.

The Effect of Antitumor Antibiotic Olivomycin A and Its New Semi-synthetic Derivative Olivamide on the Activity of Murine DNA Methyltransferase Dnmt3a.

Olivomycin A is a highly active antitumor drug that belongs to the family of aureolic acid antibiotics. The antitumor effect of olivomycin A is related to its ability to bind to the DNA minor groove in GC-rich regions as Mg2+-coordinated complexes. Characterization of cellular targets of olivomycin A and its mechanism of action is crucial for the successful application of this antibiotic in clinical practice and development of semi-synthetic derivatives with improved pharmacological properties. Previously, we have shown that minor groove ligands are able to disrupt the key epigenetic process of DNA methylation. In this paper, we have studied the impact of olivomycin A and its improved semi-synthetic analogue N,N-dimethylaminoethylamide of 1'-des-(2,3-dihydroxy-n-butyroyl)-1'-carboxy-olivomycin A (olivamide) on the functioning of de novo DNA methyltransferase Dnmt3a (enzyme that carries out methylation of cytosine residues in the DNA CG-sites in eukaryotic cells) using an in vitro system consisting of the murine Dnmt3a catalytic domain and a 30-mer DNA duplex containing four consecutive GC pairs. We have shown that olivomycin A and olivamide inhibit Dnmt3a with IC50 of 6 ± 1 and 7.1 ± 0.7 μM, respectively. Neither olivomycin A nor olivamide interfered with the formation of the specific enzyme-substrate complex; however, olivomycin A prevented formation of the covalent DNA-Dnmt3a intermediate that is necessary for the methylation reaction to proceed. The inhibitory effects of olivomycin A and olivamide can be explained by the disruption of the enzyme catalytic loop movement through the DNA minor groove (the reaction stage that precedes the covalent bond formation between DNA and the enzyme). The results of this work indicate the epigenetic contribution to the antitumor effect of aureolic acid group antibiotics.

Curcumin ameliorates PRMT5-MEP50 arginine methyltransferase expression by decreasing the Sp1 and NF-YA transcription factors in the A549 and MCF-7 cells.

The protein arginine methyltransferase 5 (PRMT5) and its catalytic partner methylosome protein MEP50 (WDR77) catalyse the mono- and symmetric di-methylation of selective arginines in various histones and non-histone target proteins. It has emerged as a crucial epigenetic regulator in cell proliferation and differentiation; which also reported to be overexpressed in many forms of cancers in humans. In this study, we aimed to assess the modulations in the expression of this enzyme upon exposure to the well-studied natural compound from the spice turmeric, curcumin. We exposed the lung and breast cancer cell lines(A549 and MCF-7) to curcumin (2 and 20μM) and observed a highly significant inhibitory effect on the expression of both PRMT5 and MEP50. The level of symmetrical dimethylarginine (SDMA) in multiple proteins, and more specifically, the H4R3me2s mark (which predominates in GC-rich motifs in nucleosomal DNA) was also diminished significantly. We also found that curcumin significantly reduced the level and enrichment of the transcription factors Sp1 and NF-YA which shares their binding sites within the GC-rich region of the PRMT5 proximal promoter. Furthermore, the involvement of both PKC-p38-ERK-cFos and AKT-mTOR signalling was observed in reducing the Sp1 and NF-YA expression by curcumin. Therefore, we propose curcumin decreased the expression of PRMT5 in these cells by affecting at least these two transcription factors. Altogether, we report a new molecular target of curcumin and further elucidation of this proposed mechanism through which curcumin affects the PRMT5-MEP50 methyltransferase expression might be explored for its therapeutic application.

Approach to Molecular Diagnosis of Chronic Granulomatous Disease (CGD): an Experience from a Large Cohort of 90 Indian Patients.

Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population. This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis. Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified. Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform. Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

How complete are “complete” genome assemblies?—An avian perspective

The genomics revolution has led to the sequencing of a large variety of nonmodel organisms often referred to as "whole" or "complete" genome assemblies. But how complete are these, really? Here, we use birds as an example for nonmodel vertebrates and find that, although suitable in principle for genomic studies, the current standard of short-read assemblies misses a significant proportion of the expected genome size (7% to 42%; mean 20±9%). In particular, regions with strongly deviating nucleotide composition (e.g., guanine-cytosine-[GC]-rich) and regions highly enriched in repetitive DNA (e.g., transposable elements and satellite DNA) are usually underrepresented in assemblies. However, long-read sequencing technologies successfully characterize many of these underrepresented GC-rich or repeat-rich regions in several bird genomes. For instance, only ~2% of the expected total base pairs are missing in the last chicken reference (galGal5). These assemblies still contain thousands of gaps (i.e., fragmented sequences) because some chromosomal structures (e.g., centromeres) likely contain arrays of repetitive DNA that are too long to bridge with currently available technologies. We discuss how to minimize the number of assembly gaps by combining the latest available technologies with complementary strengths. At last, we emphasize the importance of knowing the location, size and potential content of assembly gaps when making population genetic inferences about adjacent genomic regions.

Redox-dependent condensation of the mycobacterial nucleoid by WhiB4

Oxidative stress response in bacteria is mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered non-functional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance is unknown. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the protein’s ability to condense DNA. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of ~ 30% of genes preferentially bound by the protein, suggesting both direct and indirect effects on gene expression. We propose that WhiB4 structurally couples Mtb’s response to oxidative stress with genome organization and transcription.

Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations

BackgroundHuman cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies.ResultsThe MinION run of the HCMV (strain TB40/E) library resulted in ~ 47,000 reads from a single R9 flowcell and in ~ 100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50 = 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50 = 5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50 = 41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions.ConclusionsThird generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.

Abstract 1418: Identification of recurrent regulatory mutations in breast cancer

Abstract Since the identification of recurrent TERT promoter mutations in melanoma resulting in increased TERT expression, there has been increased interest in identifying recurrent regulatory non-coding mutations (Horn et al. 2013, Huang et al. 2013). Several studies have attempted pan-cancer analyses in order to identify these types of mutations, but often the results suffer from low coverage of regulatory regions or do not extend to breast cancer. While some breast cancer specific studies have identified some significantly mutated promoters and lncRNAs, they have often failed to incorporate transcriptome data to assess the impact and relevance of mutations on the expression of genes within tumors (Nik-Zainal et al. 2016). In order to address this, we assembled and generated a data set consisting of 458 breast cancer cases with matched tumor/normal pairs. This cohort consists of 22.4% luminal A, 19% luminal B, 16.4% HER2-enriched, 21% basal-like, 0.8% normal-like, and 20.4% unknown with regards to molecular subtype. This is important due to different breast cancer subtypes having dissimilar phenotypes and varying rates of gene coding mutations. This data set has a mix of whole genome, exome, transcriptome, and custom capture sequencing. We designed a custom capture reagent that covers regions assembled from regulatory databases, 5' untranslated regions, 500 bases upstream and downstream of transcription start sites, and 50,000 bases upstream and downstream of 178 genes that have been implicated as being important in breast cancer (Lesurf et al. 2016). While this custom capture region is similar in size to an exome, it has advantages over whole genome and exome sequencing, particularly with respect to coverage in GC-rich promoter regions. With these data, we predict that we will be able to identify novel, regulatory coding and non-coding drivers of breast cancer that would not be discovered without integrated analysis of the DNA- and RNA-seq data for each tumor. Instrument data were processed using the McDonnell Genome Institute somatic variant calling pipeline that includes 5 SNV callers and 3 indel callers. We then used these steps to filter variants: min. 20x coverage in both the tumor and normal sample, min. 2.5% tumor variant allele frequency, min. 3 variant supporting reads in the tumor sample, max. 10% variant allele frequency in the normal sample. We also filtered against gnomAD and a panel of normals. Rheinbay et al. 2017 identified recurrently mutated promoter regions for nine genes: TBC1D12, ZNF143, ALDOA, NEAT1, RMRP, CITED2, FOXA1, CTNNB1, LEPROTL1. Our preliminary analysis has revealed that we also see mutations within these regions. We plan to present on the significance of mutations within these previously seen regions, based on recurrence and transcriptome changes, as well as novel recurrent regulatory regions that our analysis reveals, particularly with respect to molecular subtype. Citation Format: Kelsy C. Cotto, Arpad Danos, Robert Lesurf, Morag Park, Malachi Griffith, Obi L. Griffith. Identification of recurrent regulatory mutations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1418.

Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region

The repressive activity of ancestral histone-like proteins helps integrate transcription of foreign genes with discrepant AT content into existing regulatory networks. Our investigations indicate that the AT-rich discriminator region located between the −10 promoter element and the transcription start site of the regulatory gene ssrA plays a distinct role in the balanced expression of the Salmonella pathogenicity island-2 (SPI2) type III secretion system. The RNA polymerase-binding protein DksA activates the ssrAB regulon post-transcriptionally, whereas the alarmone guanosine tetraphosphate (ppGpp) relieves the negative regulation imposed by the AT-rich ssrA discriminator region. An increase in the GC-content of the ssrA discriminator region enhances ssrAB transcription and SsrB translation, thus activating the expression of downstream SPI2 genes. A Salmonella strain expressing a GC-rich ssrA discriminator region is attenuated in mice and grows poorly intracellularly. The combined actions of ppGpp and DksA on SPI2 expression enable Salmonella to grow intracellularly, and cause disease in a murine model of infection. Collectively, these findings indicate that (p)ppGpp relieves the negative regulation associated with the AT-rich discriminator region in the promoter of the horizontally-acquired ssrA gene, whereas DksA activates ssrB gene expression post-transcriptionally. The combined effects of (p)ppGpp and DksA on the ssrAB locus facilitate a balanced SPI2 virulence gene transcription that is essential for Salmonella pathogenesis.

Optimizing Amplification of the GC-Rich TERT Promoter Region Using 7-Deaza-dGTP for Droplet Digital PCR Quantification of TERT Promoter Mutations

To the Editor: Droplet digital PCR (ddPCR) assays partition PCR reactions into multiple compartments. PCR amplification followed by analysis of end point fluorescence of each partition allows the absolute quantification of the target sequence. ddPCR is effective for rare mutation detection, such as in circulating tumor DNA. Recurrent mutations at 2 sites in the telomerase reverse transcriptase ( TERT )1 promoter are present in multiple malignancies. Two promoter mutations (G>A/C>T transitions occurring at chr5:1295228 or chr5:1295550, respectively, referred to as C228T and C250T) together account for over 80% of TERT promoter mutations in cutaneous melanoma (1). However, the TERT promoter has proved to be a challenging region to amplify due to its high GC content (>80%), homopolymer runs, and low sequence complexity (2). Recently, McEvoy and colleagues reported a ddPCR assay for TERT promoter mutations by use of Q solution and locked nucleic acids (3). We developed an alternative method that uses 7-deaza-2′-deoxyguanosine 5′-triphosphate (7-deaza-dGTP) as an additive for ddPCR. In contrast to McEvoy, our method analyzes the 2 mutations independently. We evaluated the use of dimethyl sulfoxide (DMSO), Q solution, and 7-deaza-dGTP as additives to improve the performance of ddPCR for the detection of TERT promoter …

Characterization of the c9orf72 GC-rich low complexity sequence in two cohorts of Italian and Turkish ALS cases

Large expansions of a noncoding GGGGCC repeat in the C9orf72 gene are the main cause of amyotrophic lateral sclerosis (ALS). The GGGGCC repeat is contiguous with another GC-rich region. Recent studies reported a significantly higher frequency of insertions/deletions within the GC-rich region in patients carrying the GGGGCC expansion. A GTGGT motif comprised within the GC-rich region, which joins two 100% GC sequences, was frequently deleted, supporting the hypothesis that these deletions could make the region more prone to slippage and pathological expansion. To confirm this hypothesis, we sequenced the GC-rich region adjacent the GGGGCC repeat in ALS patients, 116 C9orf72 expansion carriers, 219 non-carriers, and 223 healthy controls, from Italian and Turkish cohorts. Deletions were significantly more frequent in C9orf72 expansion carriers (6%) compared to non-carrier ALS patients (0.46%, OR =14.00, 95% CI =1.71–306.59, p = 0.003), to controls (0%, OR =16.29, 95% CI =2.12–725.99, p = 4.86 × 10−4) and to the whole cohort of non-carriers (0.2%, OR =28.51, 95% CI =3.47–618.91, p = 9.58 × 10−5). Among expansion carriers, deletions with or without the GTGGT motif were equally distributed (4 vs. 3). The frequency of insertions was not statistically different between C9orf72 expansion carriers and any other group including the whole cohort of non-carriers (p = 0.439, Fisher’s exact test). Our data confirmed the association between deletions within GC-rich region and the GGGGCC expansion in Italian and Turkish cases, although we did not confirm a role of the GTGGT element deletion. Further studies will be therefore necessary to assess the causal relationships between contiguous deletions of the GC-rich region and the GGGGCC expansion.

Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed ‘short acid’ (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models. We herein report that compound 3 is able to form stable complexes with DNA in the absence of Mg2+, in striking contrast to 1 whose binding to the DNA absolutely requires Mg2+. The mode of binding of 3 to DNA is similar in the presence or absence of Mg2+ as determined by circular dichroism. The affinity to DNA of 3 in Mg2+-free solution was similar to that of 1 or 3 in the presence of Mg2+ at low ionic strength. Non-electrostatic contributions to total free energy of binding of 1 and 3 to DNA were comparable for Mg2+-free complexes. Our data strongly suggest that electrostatic interaction of the positively charged 3 can compensate for the absence of divalent ions in complexes with DNA. This new property of the olivomycin A derivative expands the mechanistic knowledge of the modes of interaction with DNA of small molecular weight drug candidates.